Molecular control of gene expression in hematopoiesis and erythropoiesis is directly relevant to sickle cell anemia. To cite examples, the fetal to adult switch in globin gene expression, the development of F-cells in adult erythropoiesis, the responsiveness of hematopoietic progenitors to hemopoietins, and the expression of genes in pluripotent stem cells that are potential targets of gene therapy can all be related to sickle disease. Very little is known about the earliest molecular events in human hematopoietic cell development. Such studies are hampered by the lack of a suitable in vitro system in which to study pure, differentiating human hematopoietic stem cells. Evidence from several groups suggests that the genes for several hemopoietin receptors, including receptors for lineage- specific factors such as erythropoietin (Epo), are turned on early in hematopoiesis. Consequently, a detailed molecular understanding of the induction of such a gene might provide a window into this phase of hematopoietic cell differentiation. This proposal outlines experiments designed to open such a window by closely examining the expression of the human erythropoietin receptor (EpoR) gene in murine embryonic stem (ES) cells undergoing hematopoietic differentiation. Our central hypothesis, therefore, is that understanding the regulation of the human EpoR gene at a molecular level will teach us something new about early hematopoiesis. The proposed experiments follow up our observations that the human EpoR gene is regulated normally in differentiating ES cells; it is induced within one day of differentiation (the same as the endogenous murine EpoR gene). Furthermore, this induction is independent of GATA-1 or GATA-2 transcription factors. The proposed experiments will examine the cis regulatory elements within the human EpoR gene responsible for its induction in ES cells. Next, the factors that act in trans to effect this regulation will be identified and characterized and their functional role in hematopoiesis will be assessed. The information learned from these studies might ultimately prove valuable in understanding or treating clinical disorders such as sickle cell anemia.